The invention relates to a process for the production of a DNA (desoxyribonucleic acid) comprising the genome characteristic of that of the B hepatitis virus. It also relates to DNAs of which a fragment is constituted by a double strand DNA corresponding to that of viral B hepatitis. In addition, it relates to vectors and compositions including such DNAs, for taking advantage of their biological properties. The invention relates to a nucleic acid comprising a nucleotide sequence capable of coding an immunogenic peptide sequence corresponding to the surface antigen of the virus of viral hepatitis B, and to the polypeptides and peptides obtained. It relates also to a process enabling such a nucleic acid to be obtained.
B hepatitis is a frequent viral disease, more particularly in tropical Africa, in Southeast Asia, and in the Far East where about 10% of the people are carriers of the surface viral antigen also designated as HBs antigen.
Though the infection is often manifested by an acute form without sequelae, it can also be at the origin of a chronic hepatitis, of cirrhosis, and even of fatal hepatic necrosis. This explains the importance of studies devoted to the biology of the virus, and the recent development of a vaccine whose efficiency has been demonstrated on patients and members of the personnel of hemodialysis centers (Ph. MAUPAS, A. GOUDEAU, P. COURSAGET, J. DRUCKER and Ph. BAGROS, Intervirol., 10, 1978, p. 196-208). The Dane particle (D. S. DANE, C. H. CAMERON and M. BRIGGS, Lancet. i. 1970, p. 695-698) is at present considered as the etiological viral agent. This particle, which can be detected by observation with the electron microscope, has a diameter of 42 nm. The patient's serum in the preicteric phase contains up to 10.sup.9 or even 10.sup.10 of it per milliliter. It possesses an envelope (Australia antigen or HBs antigen), a capsid (HBc antigen), an endogenic polymerase, and a DNA molecule (J. L. Melnick, G. R. DREESMAN and F. B. HOLLINGER, Sc. Amer., 237, 1977, p. 44-52). Under observation with the electron microscope, the genome appears as a bicatenary DNA ring possessing a monocatenary region, whose length varies from one molecule to the next (J. SUMMERS, A. O'CONNELL and I. MILLIMAN, Proc. Nat. Acad. Sc., 72, 1975, p. 4597-4601), (W. S. ROBINSON, Ann. Rev. Microbiol., 31, 1977). This ring is constituted by two intertwined linear molecules of unequal lengths (as shown diagrammatically in FIG. 1). It is the smallest viral genome known in mammals. The longest strand contains about 3,200 bases. The endogen polymerase DNA can be used to repair in vitro the single strand region (b.sub.1 in FIG. 1, of the shortest strand (T. A. LANDERS, H. B. GREENBERG and W. S. ROBINSON, J. Virol., 23, 1977, p. 368-376). All these very special properties of the Dane particle further increase the interest of studying the biology of this virus.
Electrophoretic analysis of the proteins of the envelope has shown the presence of 2 to 7 polypeptides of which the principal are called: polypeptide I and polypeptide II (PETERSON D. L., ROBERS I. M. and VYAS G. N. (1977) Proc. Nat. Acad. Sci., USA, 74 1530-1534, and PETERSON D. L., CHIEN D. Y., VYAS G. N., NITECHKI D., and BOND H. (1978). In Viral Hepatitis, ed. G. VYAS, S. COHEN and R. SCHMID, The Franklin Institute Press, Philadelphia, 569-573).
The Polypeptide I has a weight of 22,000 to 26,000 daltons. Polypeptide II is glycosylated and has a molecular weight of 28,000 to 30,000 daltons. The amino acid composition of these two polypeptides is very similar, the sequences which form, respectively, their 15 first amino acids (from the N-terminal end) and their last 3 amino acids are identical, so that the hypothesis has formulated that polypeptide II could differ from polypeptide I only by a glycosylation. Until now, the sequence of the I and II polypeptides themselves, and the location in the viral DNA of the sequence coding these peptides have not been done.
Study of the virus is, however, at present made particularly difficult by reason of the difficulties of supplies of serum containing Dane particles. Even a rich serum does not permit the preparation of large amounts of DNA (of the order of 1 .gamma. of DNA per volume of 500 ml of serum). It is hence necessary to collect: serums of various origins corresponding to several genetic variants (J. P. SOULIER and A. M. COUROUCE-PAUTY, Vox Sang. 25, 1973, p. 212-235), which renders precarious a study of the primary structure of the genome. The presence of the single strand region makes difficult, moreover, the establishment of a physical map by restriction enzymes.
The problem of the isolation of relatively large amounts of viral particles attains a still increased importance, when it is desired to have available sufficient amounts of viral particles, more particularly of their HBs antigens, which appear to carry a surface antigen (protein) having vaccinating properties. The present methods of vaccination, if they have demonstrated their efficiency, are not however absolutely devoid of drawbacks. In particular, preparations of HBs, used as a vaccine, may contain antigen components coming from hepatic cells, which can be the origin of an autoimmune response (B. S. BLUMBERG, Science, 197, 1977, p. 17-24).
Study of the virus is also extremely difficult to the extent that no cell culture system is available enabling the propagation of the virus. This difficulty has already in part been ovecome, more particularly as regards the ayw serotype. The whole DNA (genome) of the virus has been identified and cloned, notably in E. coli, after its previous insertion in the single EcoRI site of a .lambda. gt. WES. .lambda.B vector, according to the technique by FRITSCH A., POURCEL C., CHARNAY P., and TIOLLAIS P. (1978) C. R. Acad. de Paris, 287, 1,453-456).
Until now, the sequence of the I and II polypeptides themselves, and the location in the viral DNA of the sequence coding these peptides have not been done.
It is, therefore, an object of the invention notably to overcome these difficulties, more particularly to provide a process enabling the production of DNA of B hepatitis virus (or of the Dane particle), in sufficient amounts for the realization of the above-mentioned studies, and in a state of purity such that its use can be contemplated, even for therapeutic uses.
It is also an object of the invention to provide a much smaller DNA sequence than the viral DNA itself, containing the sequence adapted to code the peptide sequence endowed with immunogenic properties enabling, when it is introduced into the organism of a living host, to induce the formation by the latter of antibodies capable of protecting this same host subsequently with respect to the virus of viral hepatitis B, notably when the latter is in virulent state.